Among filamentous fungi, in particular, Aspergillus including Aspergillus oryzae (yellow Aspergillus) etc. has been traditionally used in brewing industry in Japan for producing sake, bean paste, soy sauce, mirin, and the like, and directly eaten. Aspergillus is a safe source of genes listed up in GRAS (Generally Recognized as Safe) by US FDA (Food and drug Administration).
Therefore, in the safety test such as a chronic toxicity test required when genes derived from general fungi are used for foods, etc., in the case of genes derived from general fungi, the cost is about 1 billion yen. On the other hand, in the case of genes that are the above-mentioned GRAS genes, it is advantageous that the cost can be reduced to about one-third of the cost and further that it takes a shorter time to conduct the test as compared with the case of general genes.
Thus, filamentous fungi, in particular, Aspergillus could provide a source of genes with a high utility value from the safe and economical viewpoint.
Therefore, by clarifying information on the genome DNA of these fungi and clarifying the functions of genes encoded thereby, it is possible to provide an effective using method of safe gene resources, for example, production of materials with the use of biotechnology, in food industry; and to provide useful information for screening various kinds of genes in the field of agricultural chemicals and medicine.
Furthermore, it would provide a useful tool for analyzing genome information of grain contamination fungi such as closely-related species including, for example, Aspergillus flavus, Aspergillus fumigatus, etc. and human-infecting bacteria.
As a result of investigation under the above-mentioned circumstances, the present inventors have succeeded in analyzing the genome of Aspergillus oryzae, that is, a kind of Aspergillus and have determined the base sequence thereof (and an amino acid sequence encoded by the base sequence) and various functions, etc. Based on such results, the present inventors disclosed various DNAs derived from Aspergillus oryzae, as well as a primer set for amplifying a gene of filamentous fungi in GRAS grade including a nucleotide sequence prepared by these DNAs and a probe for detecting genes of filamentous fungi, etc. in the prior patent application (Japanese Patent Application No. 2001-403261).
The present inventors have conducted a further investigation based on the resultant genome information on Aspergillus. That is to say, the present inventors paid attention to phospholipase A2 and identified a sequence encoding phospholipase A2 from the resultant base sequences. The present inventors have also tried to identify an amino acid sequence of a protein encoded by the sequence. Note here that phospholipase A2 is an enzyme for hydrolyzing the ester bond at the 2 position of glycerophospholipid so as to separate between fatty acid and lysophospholipid. The presence of phospholipase A2 derived from animals has been traditionally known and such phospholipase A2 has been widely applied in food industry including manufacture of lysolecithin as an emulsifying agent (see, for example, Japanese Patent Unexamined Publication No. H10-042884), a degumming step of fats and oils (see, for example, Industrial Enzymology 2nd ed., p 299-300 (1996)), breadmaking (see, for example, Japanese Patent Unexamined No. S60-2135), functional phospholipid (see, for example, Oleo Science Vol. 2, No. 1 (2002)), and the like. Furthermore, it can be used for development of anti-inflammatory agents, and therapeutic agents for septicemia, rheumatism, asthma, ischemic disease, ischemic reperfusion disorder, and the like. Recently, however, enzymes derived from animals have been tended to be avoided by consumers and food manufacturing companies due to their origins. Enzymes derived from microorganisms that are regarded as being safer have been demanded. In recent years, phospholipase A2 derived from microorganism has been studied, an enzyme of Streptomyces violaceoruber that is a kind of Streptomyces has been found (see Japanese Patent Unexamined Publication No. 6-327468). In more recent years, an enzyme derived from Tuber borchii that is a kind of filamentous fungi (see Soragni et al., The EMBO Journal, 20 (18) 5079-5090 (2001)), and an enzyme derived from filamentous fungi of Helicosporium sp. (Wakatsuki et al., Biochin. Biophys. Acta 1522, 74, (2001)) have been reported. However, these microorganisms have never been used for foods and therefore, enzymes derived from safer fungi have been demanded.
It is therefore an object of the present invention to provide phospholipase A2 derived from Aspergillus and DNA encoding phospholipase A2 as well as a method for producing phospholipase A2 derived from Aspergillus, and the like.